Method for producing L-methionine by fermentation

Abstract

L-Methionine is produced by culturing a microorganism which is deficient in repressor of L-methionine biosynthesis system and/or enhanced intracellular homoserine transsuccinylase activity is cultured in a medium so that L-methionine should be produced and accumulated in the medium, and collecting the L-methionine from the medium. The microorganism preferably further exhibits reduced intracellular S-adenosylmethionine synthetase activity, L-threonine auxotrophy, enhanced intracellular cystathionine γ-synthase activity and enhanced intracellular aspartokinase-homoserine dehydrogenase II activity. The present invention enables breeding of L-methionine-producing bacteria, and L-methionine production by fermentation.

1. A method for producing L-methionine which comprises culturing a recombinant  Escherichia  bacterium in a medium to produce and accumulate L-methionine in the medium in an amount in excess of the corresponding unmodified  Escherichia  bacterium, and collecting the L-methionine from the medium, wherein
 the bacterium is deficient in repressor of L-methionine biosynthesis system encoded by the endogenous metJ gene and has L-methionine productivity, 
 activity of intracellular homoserine transsuccinylase encoded by the metA gene of a  Escherichia  bacterium is increased compared to an unmodified  Escherichia  bacterium by increasing copy number of the metA gene including its own promoter, or replacing the native promoter with a stronger promoter, and 
 the bacterium comprises at least one characteristic selected from the group consisting of: 
 (a) exhibits reduced activity of intracellular S-adenosylmethionine synthetase encoded by the endogenous metK gene as compared to an unmodified  Escherichia  bacterium; 
 (b) exhibits L-threonine auxotrophy; 
 (c) exhibits enhanced activity of intracellular cystathionine γ-synthase encoded by the metB gene of a  Escherichia  bacterium and enhanced activity of intracellular aspartokinase-homoserine dehydrogenase II encoded by the metL gene of a  Escherichia  bacterium as compared to an unmodified  Escherichia  bacterium by increasing copy number of each of the genes including their own promoters, or replacing the native promoter with a stronger promoter; and 
 (d) has a homoserine transsucinylase for which concerted inhibition by L-methionine and S-adenosylmethionine is desensitized, wherein the homoserine transsuccinylase comprising the amino acid sequence of SEQ ID NO: 26 contains at least one amino acid replacement wherein said at least one amino acid replacement is independently selected from the group consisting of replacement of the amino acid residue Arg-27 with cysteine, replacement of the amino acid residue Ile-296 with serine, and replacement of the amino acid residue Pro-298 with leucine. 
 
     
     
       2. The method according to  claim 1 , wherein the bacterium is  Escherichia coli.    
     
     
       3. The method according to  claim 1 , wherein the bacterium comprises at least the characteristic (a). 
     
     
       4. The method according to  claim 1 , wherein the bacterium comprises at least the characteristic (b). 
     
     
       5. The method according to  claim 1 , wherein the bacterium comprises at least the characteristic (c). 
     
     
       6. The method according to  claim 1 , wherein the bacterium comprises at least the characteristic (d). 
     
     
       7. The method according to  claim 1 , wherein the bacterium comprises the characteristics (a) and (b). 
     
     
       8. The method according to  claim 1 , wherein the bacterium comprises the characteristics (a) and (c). 
     
     
       9. The method according to  claim 1 , wherein the bacterium comprises the characteristics (a) and (d). 
     
     
       10. The method according to  claim 1 , wherein the bacterium comprises the characteristics (b) and (c). 
     
     
       11. The method according to  claim 1 , wherein the bacterium comprises the characteristics (b) and (d). 
     
     
       12. The method according to  claim 1 , wherein the bacterium comprises the characteristics (c) and (d). 
     
     
       13. The method according to  claim 1 , wherein the bacterium comprises the characteristics (a), (b), and (c). 
     
     
       14. The method according to  claim 1 , wherein the bacterium comprises the characteristics (a), (b), and (d). 
     
     
       15. The method according to  claim 1 , wherein the bacterium comprises the characteristics (a), (c), and (d). 
     
     
       16. The method according to  claim 1 , wherein the bacterium comprises the characteristics (b), (c), and (d). 
     
     
       17. The method according to  claim 1 , wherein the bacterium comprises the characteristic (a), (b), (c), and (d). 
     
     
       18. The method according to  claim 1 , wherein the activity of intracellular S-adenosylmethionine synthetase is reduced due to that the bacterium has S-adenosylmethionine synthetase which contains amino acid substitution which is selected from the group consisting of replacement of the amino acid residue Ile-303 with leucine, replacement of the amino acid residue Val-185 with glutamic acid, and replacement of amino acid residues 378-384 with the amino acid sequence of SEQ ID NO: 29, respectively in the amino acid sequence of SEQ ID NO: 18. 
     
     
       19. The method according to  claim 3 , wherein the activity of intracellular S-adenosylmethionine synthetase is reduced due to that the bacterium has S-adenosylmethionine synthetase which contains amino acid substitution which is selected from the group consisting of replacement of the amino acid residue Ile-303 with leucine, replacement of the amino acid residue Val-185 with glutamic acid, and replacement of amino acid residues 378-384 with the amino acid sequence of SEQ ID NO: 29, respectively in the amino acid sequence of SEQ ID NO: 18. 
     
     
       20. The method according to  claim 1 , wherein the L-threonine auxotrophy is due to deletion of the thrBC genes. 
     
     
       21. The method according to  claim 4 , wherein the L-threonine auxotrophy is due to deletion of the thrBC genes.